Difference between revisions of "Part:BBa K2551003"

Latest revision as of 00:58, 19 October 2021

fruA exo-beta-D-fructosidase - CDS

This encodes one type of Fructosidase found in Streptococcus mutans UA159 (strain: UA159)

EDIT (Team ULaval 2020, 2020-10-20): The annotations for parts BBa_K2551002 and BBa_K2551003 were inverted. Part BBa_K2551003 encodes one particular dextranase found in Streptococcus mutans UA159 (strain: UA159). This kind of enzyme decompose dextran under certain condition. Validated by translating and BLASTing the sequence.

EDIT (Team ULaval 2021, 2021-10-18): We expressed this dextranase in a pET28a vector using Escherichia coli BL21 and purified it using IMAC chromatography. We observed signs of degradation, which were more important at 18 °C. As Kim et al., 2011 demonstrated, the amino residue fragment from 100 to 731(lower band at 69.4kDa) is sufficient to perform the dextranase activity of the enzyme whilst reducing the sensitivity to proteolytic activity. For a future expression and purification experiment, we would suggest to the next team to use this part to modify its sequence slightly by conserving only those residues. This truncation of the protein will provide a homogeneous protein extract and allow the team to use the dextranase without worrying about its different forms. For more information, see the experience tab of this part or see our 2021 wiki https://2021.igem.org/Team:ULaval/Results.

Kim, Y.-M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M.-S., Fujimoto, Z., Funane, K., Kim, D., & Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied Microbiology and Biotechnology, 91(2), 329–339.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 856
Illegal BsaI.rc site found at 1669
Illegal BsaI.rc site found at 2200

@@ Line 7: / Line 7: @@
 EDIT (Team ULaval 2020, 2020-10-20): The annotations for parts BBa_K2551002 and BBa_K2551003 were inverted. Part BBa_K2551003 encodes one particular dextranase found in Streptococcus mutans UA159 (strain: UA159). This kind of enzyme decompose dextran under certain condition. Validated by translating and BLASTing the sequence.
-EDIT (Team ULaval 2021, 2021-10-18): We expressed this dextranase in a pET28a vector using Escherichia coli BL21 and purified it using IMAC chromatography. We observed signs of degradation, which were more important at 18 °C. As Kim et al., 2011 demonstrated, the amino residue fragment from 100 to 731(lower band at 69.4kDa) is sufficient to perform the dextranase activity of the enzyme whilst reducing the sensitivity to proteolytic activity. For a future expression and purification experiment, we would suggest to the next team to use this part to modify its sequence slightly by conserving only those residues. This truncation of the protein will provide a homogeneous protein extract and allow the team to use the dextranase without worrying about its different forms.
+EDIT (Team ULaval 2021, 2021-10-18): We expressed this dextranase in a pET28a vector using Escherichia coli BL21 and purified it using IMAC chromatography. We observed signs of degradation, which were more important at 18 °C. As Kim et al., 2011 demonstrated, the amino residue fragment from 100 to 731(lower band at 69.4kDa) is sufficient to perform the dextranase activity of the enzyme whilst reducing the sensitivity to proteolytic activity. For a future expression and purification experiment, we would suggest to the next team to use this part to modify its sequence slightly by conserving only those residues. This truncation of the protein will provide a homogeneous protein extract and allow the team to use the dextranase without worrying about its different forms. For more information, see the experience tab of this part or see our 2021 wiki https://2021.igem.org/Team:ULaval/Results.
 Kim, Y.-M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M.-S., Fujimoto, Z., Funane, K., Kim, D., & Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied Microbiology and Biotechnology, 91(2), 329–339.